The X,K-ATPases represent a family of structurally related but functionally distinct iontransporting ATPases including receptors for cardiac glycosides (Na,K-ATPase) and anti-ulcer drugs (gastric H,K-ATPase). Studies proposed in this project focus on the molecular aspects of the human nongastric H,K-ATPase, and the muscle-specific beta-m-protein, which is a novel memberof the X,K-ATPase family. Nongastric H,K-ATPase plays an important role in the maintenance of electrolyte homeostasis under pathophysiological conditions. Most significantly, this human ion pump is sensitive to cardiac glycosides, particularly to digoxin and digitoxin, which are widely used in clinical practice. During the previous funding period we have characterized basic ion-transport and catalytic functions of recombinant human nongastric H,K-ATPase via expression in Xenopus oocytes and insect cells. The objective of the first group of proposed studies is to define specific features of reaction mechanism of nongastric H,K-ATPase (Specific Aim 1) and to examine the roles of particular structural motifs and residues in the determination of ion specificity and inhibitor sensitivity of human nongastric H,K-ATPase (Specific Aim 2). The anticipated results should provide new insight into molecular basis of the human nongastric H,K-ATPase functioning and define relationship between members of X,K-ATPase family. The results of our preliminary studies demonstrated that recently identified muscle-specific betam protein exhibits structural features, cellular location and functional roles all of which are clearly different from those of the other X,K-ATPase beta subunits. The unique temporospatial expression pattern suggests that beta-m may play an essential role in development of heart and skeletal muscle. The hypothesis to be tested is that betam is associated in vivo with the protein(s) different from known X,K-ATPase alpha-subunits, thus being a component of an unknown system, which might be an unidentified muscle-specific PATPase. The main objective of Specific Aim 3, is to identify protein counterpart(s) of the muscle specific beta-m. These experiments should clarify the relationship between the beta-m and other proteins in skeletal muscle and heart and provide insight into understanding the beta-m physiological function.